Amiya K. Hajra

Phenotype of patients with peroxisomal disorders subdivided into sixteen complementation groups

OBJECTIVE To use the technique of complementation analysis to help define genotype and classify patients with clinical manifestations consistent with those of the disorders of peroxisome assembly, namely the Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). STUDY DESIGN Clinical findings, peroxisomal function, and complementation groups were examined in 173 patients with the clinical manifestations of these disorders. RESULTS In 37 patients (21%), peroxisome assembly was intact and isolated deficiencies of one of five peroxisomal enzymes involved in the beta-oxidation of fatty acids or plasmalogen biosynthesis were demonstrated. Ten complementation groups were identified among 93 patients (54%) with impaired peroxisome assembly and one of three phenotypes (ZS, NALD, or IRD) without correlation between complementation group and phenotype. Forty-three patients (25%) had impaired peroxisome assembly associated with the RCDP phenotype and belonged to a single complementation group. Of the 173 patients, 10 had unusually mild clinical manifestations, including survival to the fifth decade or deficits limited to congenital cataracts. CONCLUSIONS At least 16 complementation groups, and hence genotypes, are associated with clinical manifestations of disorders of peroxisome assembly. The range of phenotype is wide, and some patients have mild involvement.

Deficiency of enzymes catalyzing the biosynthesis of glycerol-ether lipids in Zellweger syndrome. A new category of metabolic disease involving the absence of peroxisomes.

The Zellweger cerebro-hepato-renal syndrome is a genetic disease characterized by the absence of peroxisomes and deficiency of glycerol-ether lipids in several tissues. We measured the activity of dihydroxyacetone phosphate (DHAP) acyltransferase, a peroxisomal enzyme with a major role in ether lipid synthesis, in fibroblasts and leukocytes from patients with Zellweger syndrome. Control skin and amniotic-fluid fibroblasts had normal activity of DHAP acyltransferase (0.28 to 0.3 nmol per minute per milligram of protein), whereas fibroblasts from three patients with Zellweger syndrome had deficient activity (0.013 +/- 0.006 nmol per minute per milligram of protein). The activity of the enzyme in leukocytes and levels of plasmalogens (the major class of cellular glycerol-ether lipids) in erythrocytes were also deficient in a patient, but normal levels of leukocyte enzyme and erythrocyte plasmalogens were found in her parents. Other enzymes of the acyl DHAP pathway exhibited alterations in fibroblasts from patients with Zellweger syndrome, and the activity of the glycerophosphate acyltransferase was also reduced. These results support prior studies emphasizing the role of peroxisomes and the acyl DHAP pathway in cellular ether lipid synthesis, establish Zellweger syndrome cells as valuable for elucidating peroxisomal functions, and provide prenatal and postnatal diagnostic assays as well as potential therapeutic strategies for Zellweger syndrome.

Acyl dihydroxyacetone phosphate: Precursor of alkyl ethers

Abstract 1-0-Alkyl dihydroxyacetone phosphate is biosynthesized in guinea pig liver mitochondria and in rat brain microsomes by direct reaction of a long chain alcohol and acyl dihydroxyacetone phosphate. ATP and Mg ++ are stimulatory but coenzyme A is not necessary for the reaction.

Biosynthesis of alkyl-ether containing lipid from dihydroxyacetone phosphate

Abstract A lipid, stable to alkaline methanolysis, is formed when liver mitochondria or brain microsomes are incubated with dihydroxyacetone phosphate, long chain alcohol, ATP, CoA, NaF and Mg ++ . The lipid has been identified as alkyl dihydroxyacetone phosphate. This lipid is reduced by NADPH to 1-alkyl glycerol-3-phosphate and is further acylated to form 1-alkyl 2-acyl glycerol-3-phosphate.

On extraction of acyl and alkyl dihydroxyacetone phosphate from incubation mixtures

Chloroform-methanol mixture was shown to extract acyl and alkyl dihydroxyacetone phosphate from enzyme incubation mixtures. However, when the lipid extract was washed with water to remove nonlipid materials, 70–80% of acyl and alkyl dihydroxyacetone phosphate were lost in the aqueous phase. It was shown that, keeping the pH low (<2.5) during the partition of lipids by the Bligh and Dyer method, most (>95%) of the acyl and alkyl dihydroxyacetone phosphate were recovered in the chloroform-rich phase. n-Butanol was shown to extract 80–90% of these lipids from incubation mixtures at neutral pH.

Quantification, Characterization and Fatty Acid Composition of Lysophosphatidic Acid in Different Rat Tissues

The amount and composition of lysophosphatidate present in different rat tissues have been estimated by an internal standard method in which a synthetic unnatural isomer (1-heptadecanoyl-rac-glycerol-3-phosphate) was added to the total lipid extracts, and the fatty acid composition of purified lysophosphatidate was determined. Lipids from tissues were extracted under acidic conditions, and the lysophosphatidate was purified by solvent partitions followed by thin-layer chromatography in multiple solvent systems. The purified lipid was shown to be 1-acyl-sn-glycerol-3-phosphate by chromatographic and chemical analysis, by its resistance to hydrolysis when treated with phospholipase A2 and also by its complete conversion to 1-acyl-sn-glycerol when treated with alkaline phosphatase. The fatty acid consituents of this lipid were determined by gas-liquid chromatography of the derived methyl esters. The concentrations (nmol/g of tissue) of lysophosphatidate in various tissues were: 86.2±4.2 in brain, 60.3±6.3 in liver, 46.4±6.5 in kidney, 30.6±5.0 in testis, 22.3 in heart and 19.3 in lung. Mostly (80%) saturated fatty acids were found to be present in this lyso lipid. A significantly high level of stearic acid was present in this lipid from all the tissues (50–60% in liver, kidney, brain and testis, and about 40% in heart and lung) compared to plamitic acid (10–15% in liver, kidney and brain and 25–30% in testis, heart and lung). The fatty acid compositions of phosphatidic acid, the putative product of lysophosphatidate acylation, from different tissues were also determined and palmitate was found to be the major saturated fatty acid. These results suggest that tissue lysophosphatidic acid is not only formed byde novo biosynthesis but is also generated via the breakdown of phospholipids such as phosphoinositides.

A Method for the Chemical Synthesis of 14C-Labeled Fatty Acyl Coenzyme A's of High Specific Activity

A simple and reliable method, based on that described by W. Seubert (1960, Biochem Prep. 7, 80-83), has been developed for the chemical synthesis of radioactive acyl coenzyme A’s. I-14C-labeled fatty acids (palmitic, oleic, and linoleic) are converted to their acyl chlorides with oxalyl chloride. The [l-14C]acyl chlorides are then condensed with a two- to three-fold molar excess of coenzyme A in a bicarbonate-buffered tetrahydrofuran solution to form the corresponding [l-14C]acyl coenzyme A’s. The overall yields are near 75%, and the purities are greater than 90% based on spectral, chromatographic, and enzymatic properties. During the course of studying a very lowactivity enzyme system in brain which utilizes [14C]acyl CoA as substrate, it became necessary to synthesize this labeled compound at a very high specific activity. A number of chemical and enzymatic methods have been described for the preparation of acyl CoA (1-9). However, none of the procedures was suitable for our purpose. Most of the chemical methods require an excess of the fatty acid derivative to esterify limiting amounts of CoASH, with an unacceptably low conversion of the costly radioactive fatty acid to product. In the enzymatic methods, utilizing acyl-CoA synthetase, the yields are low (60%) and the product is probably contaminated by other lipids (1,2) unless lengthy procedures are first employed to purify the enzyme (3). We have successfully used the procedure originally described by Seubert (4,5) to prepare nonradioactive acyl CoA, but when the procedure was scaled down for the preparation of radioactive acyl CoA and excess acyl chloride was not used, yields became low and erratic. Some modifications were therefore necessary to prepare radioactive acyl CoA. We have examined the conditions for the synthesis of acyl CoA in detail with the objective of obtaining a high rate of conversion of radioactive fatty acids to acyl CoA’s. By the use of an excess of CoA and by the inclusion of a bicarbonate buffer for pH control, we have developed a method for the preparation of high specific activity [lJ4C]acyl CoA’s, both saturated and unsaturated, with reproducible yields. The reaction conditions and detailed methods are described here.

Dietary ether lipid incorporation into tissue plasmalogens of humans and rodents.

Chronic feeding of 1-O-octadecyl-sn-glycerol (batyl alcohol) to patients suffering from congenital deficiency in tissue ether glycerolipids showed an increase in the plasmalogens content of their erythrocytes. However, nothing is known about the ether lipid content of other tissues in these patients. Feeding 1-O-heptadecyl-sn-glycerol to young rats showed that this uncommon ether lipid was incorporated to a high extent into the plasmalogens of all tissues except brain. Comparative studies with other precursors, such as 3-O-heptadecyl-sn-glycerol, heptadecanol and heptadecanoic acid, indicated a stereospecific incorporation of the dietary 1-O-alkyl-sn-glycerols into tissue plasmalogens without cleavage of the ether bond. Dietary ether lipids were also shown to be transferred from mothers to suckling rats, but not from pregnant rats to fetuses. The implication of these results to possible dietary ether lipid therapy for patients suffering from peroxisomal disorders is discussed.

Zellweger Syndrome: Biochemical and Morphological Studies on Two Patients Treated with Clofibrate

ABSTRACT: Two infants with Zellweger syndrome (cerebro- hepato-renal syndrome) have been studied biochemically and morphologically. Peroxisomal enzymes involved in respiration, fatty acid β-oxidation, and plasmalogen biosynthesis were assessed. In liver, catalase was present in normal amounts but was located in the cell cytosol. Dihydroxyacetone phosphate acyltransferase activity was less than one-tenth of normal. The amount of the bifunctional protein catalyzing two β-oxidation reactions was found by immunoblotting to be greatly reduced. Catalase activity was normal in intestine. D-Amino acid oxidase was subnormal in kidney. The observed enzyme deficiencies may plausibly explain many of the metabolite imbalances observed clinically. Morphologically, peroxisomes were absent from liver. In intestine, normal peroxisomes were also missing, but some rare, smaller (0.04–0.13μm) bodies were seen with a slight positive cytochemical reaction for catalase. These results, together with current concepts of peroxisome biogenesis, suggest but do not prove, that the primary defect in Zellweger syndrome may be in peroxisome assembly. The infants were treated with clofibrate, but it was ineffectual as assessed biochemically, morphologically, and clinically.

MOLECULAR SPECIES OF DIACYLGLYCEROLS AND PHOSPHOGLYCERIDES AND THE POSTMORTEM CHANGES IN THE MOLECULAR SPECIES OF DIACYLGLYCEROLS IN RAT BRAINS

Abstract: The molecular species of 1,2‐diacyl‐sn‐glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4‐phosphate (PIP), and phosphatidylinositol 4,5‐bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse‐phase HPLC. The total amount and the concentration of each species were quantified by using 1,2‐distearoyl‐sn‐glycerol (18:0–18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn‐1 and sn‐2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0–20:4 (35%), 16:0–18:1 (15%), 16:0–16:0 (9%), and 16:0–20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse‐phase HPLC, PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0–20:4 species (50% in PI and ∼65% in PIP and PIP2). PS contained mainly the 18:0–22:6 (42%) and 18:0–18:1 (24%) species. PE was mainly composed of the 18:0–20:4 (22%), 18:0–22:6 (18%), 16:0–18:1 (15%), and 18:0–18:1 (15%) species. In PC the main molecular species were 16:0–18:1 (36%), 16:0–16:0 (19%), and 18:0–18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40–50 nmol/g in 0 min to 210–290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven‐ to 10‐fold) were found for the 18:0–20:4 and 16:0–20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.